The neuronal synapse is a complex plasma membrane housing thousands of proteins whose aggregate functions define cognition, the processing of sensory information, and motor control. In Alzheimer's disease (AD), the synaptic targeting of toxins known as Amyloid 2-Derived Diffusible Ligands (ADDLs) is thought to induce synapse loss and other pathological processes. Previous work in the Klein lab and other labs suggests that ADDL targeting is specific and receptor-mediated - binding synaptic sites in a tissue-, cell-, and synapse-specific manner. However, competing hypotheses regarding the mechanism of ADDL association with neurons are also supported in the literature. Currently, the lack of direct mechanistic insight into neural ADDL binding is a significant barrier to progress in studying the pathways initiated by ADDL binding and designing therapeutic strategies to prevent neuronal ADDL targeting. We will employ novel, cell-free strategy to test the receptor-mediated hypothesis of ADDL binding by engineering soluble membrane protein libraries isolated from neural membranes. These libraries are created by transferring membrane components from heterogeneous biological membranes into nanodiscs - nanoscale, soluble domains of lipid bilayers encircled by a ring of stabilizing proteins. Nanodiscs allow the controlled incorporation and stabilization of one to several membrane proteins, and their use as an in vitro model system for studying ADDL binding represents an enabling technology allowing the application of biochemical and biophysical tools to gather direct, molecular-level information on the nature of neuronal ADDL association. 7 In Aim 1, we will determine the range of molecular interactions between ADDLs and constituents of neural membranes, testing the hypothesis that ADDLs attack neurons through specific toxin receptors. To enable this analysis, we will develop and characterize soluble, nanodisc-based, membrane protein libraries that recapitulate neuronal ADDL binding as evaluated by a number of hallmarks including binding specificity between cell populations and the ability to affect ADDL/nanodisc binding using known modulators. 7 In Aim 2, as an initial therapeutic application for an in vitro ADDL binding assay, we will develop and employ a cell-free high-throughput assay for potential AD therapeutics. The proposed assays evaluate the ability of small molecules to inhibit the interaction of ADDLs with nanodiscs containing ADDL binding sites isolated from biological membranes. Successful compounds will be validated in cell-based assays for ADDL binding and toxicity. The nanodisc-encapsulation of membrane protein libraries proposed here represents a novel solution to the perennial problems of membrane protein insolubility and synaptic membrane complexity, allowing progress in outlining the mechanistic basis of ADDL-induced synaptic pathology in AD.